DNA methylation is required for normal development of higher eukaryotes, presumably in part because of its role in genome defense, genomic imprinting and in other silencing processes such as X-chromosome inactivation. Abnormal DNA methylation has been implicated in tumorigenesis, imprinting disorders, Fragile-X syndrome, diabetes and neurologic disorders (e.g. bipolar syndrome, Alzheimer's, autism and schizophrenia). DNA methylation is dispensable in the fungus Neurospora crassa, facilitating genetic studies. The overall goal of the proposed research is to elucidate the mechanism of DNA methylation in eukaryotes by taking advantage of this outstanding model system. The work will be facilitated by valuable resources and tools including: 1. Efficient procedures for gene targeting and epitope-tagging; 2. A collection of gene knockout strains covering ~80% of the genome; 3. The mutagenic process, RIP (repeat-induced point mutation), for reverse-genetics; 4. Efficient high throughput sequencing coupled with bisulfite methylation analysis, chromatin immunoprecipitation and DamID methodology; 5. Sensitive proteomic (mass spectrometry) and immunological methods for identification of proteins and their modifications. An important goal of the proposed research is to identify all the components of the DNA methylation machinery and to elucidate their interrelationships. It is important to determine how sequences to be methylated are targeted and to expand our mechanistic understanding of the downstream steps required for heterochromatin formation and DNA methylation (Aims 1 & 2). Another general goal of the research is to advance our understanding of the regulation of DNA methylation by identifying and characterizing the mechanisms that modulate methylation patterns (Aim 3). A final goal is to improve our understanding of mechanisms that work alongside DNA methylation to silence sequences (Aim 4). Specific aims of the project are: 1. To exploit new and existing dim (defective in methylation) mutants to elucidate the mechanism of DNA methylation. 2. To test elements of heterochromatin for roles in the establishment and/or maintenance of silencing. 3. To characterize modifiers of DNA methylation and silencing. 4. To characterize silencing mutants that do not show DNA methylation defects.